Pharmaceutical applications of hyaluronic acid preparations

ABSTRACT

The invention relates to novel applications of pharmaceutical compositions containing preferably long-chain hyaluronic acids which are cross-linked or not cross-linked, and conventional adjuvants and/or supporting materials.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application of PCT/EP02/12659,filed May 22, 2003, which claims priority to foreign applicationsDE10155440.1, filed Nov. 12, 2001, and DE10209966.9, filed Mar. 7, 2001.

The present invention relates to novel applications of pharmaceuticalcompositions which comprise crosslinked or uncrosslinked and preferablylong-chain hyaluronic acid as well as customary pharmaceutical auxiliaryand/or carrier substances. One aspect relates to the use of acomposition which comprises hyaluronic acid associated with heparin forreleasing heparin in a delayed manner, for example in connection withtreating wounds, scars and keloids, for inhibiting blood coagulation orfor relieving pain. Another aspect of the invention relates to the useof a hyaluronic acid-comprising pharmaceutical composition for treatingviral infections, in particular for treating an infection withherpesviruses. Yet another aspect of the invention relates to the use ofa hyaluronic acid-comprising pharmaceutical composition as an analgesic,in particular for administration in the region of nerve endings.Finally, yet another aspect of the invention relates to the use of ahyaluronic acid-comprising pharmaceutical composition for tauteningskin, in particular for reducing the size of wrinkles in the facialregion in a long-lasting manner.

Hyaluronic acid is an unsulfated glycosaminoglycan which is found in thehuman body in synovial fluid and in extracellular matrices. It isfrequently used as a building block for biocompatible and biologicallydegradable polymers in a variety of medical applications.

European patent 0619737 relates to a pharmaceutical composition for thenontopical treatment of wounds, scars and keloids, which compositioncomprises one or more crosslinked glycosaminoglycan(s) and customarypharmaceutical auxiliary and/or carrier substances. Hyaluronic acid andheparin are mentioned, inter alia, as being examples ofglycosaminoglycans. The publication also discloses the combination ofcrosslinked glycosaminoglycans and other pharmaceutical active compoundssuch as antibiotics.

It has now been found, surprisingly, that a pharmaceutical compositionwhich comprises crosslinked or uncrosslinked hyaluronic acid, preferablylong-chain hyaluronic acid, associated with heparin, preferably withshort-chain heparin which has a chain length of, for example, from 5 to10 saccharide units, is suitable for releasing heparin in a delayedmanner, for example for local applications, such as for treating wounds,scars or keloids, or for relieving pain, and also for systemicapplications, such as inhibiting blood coagulation. In addition to itsown advantageous physiological effects, the hyaluronic acid serves as amatrix for enabling heparin to be released in a controlled manner. Therate of release can be controlled by way of the degree of crosslinking,and the nature of the crosslinking, of the hyaluronic acid as well asthe nature of the heparin and its association with the hyaluronic acid.

The composition according to the invention has an inhibitory effect onkeloid formation, particularly when it is administered nontopically(within the lesion). Administration of the compositions according to theinvention can be used to successfully treat all types of scar, includingdeep scar formations in connective tissue, such as Dupuytren's diseaseof the palmar surfaces or what is termed plastic induration of thepenis, which develop without any preceding trauma (cross-section).

The composition exhibits a number of advantages as compared with knownpreparations. Thus, it is possible to administer it by injection in amanner which is to a large extent pain-free. In addition, no localhyperintensive reactions and no unwanted systemic side-effects occur.Another advantage is its biological degradability in the body. Anadvantage as compared with the compositions disclosed in European patent0 619 737 is, in particular, that the rate at which the heparin isreleased can be varied in dependence on the application, e.g. inaccordance with the nature of the hyaluronic acid matrix employed and ofthe heparin employed. Thus, it is possible to achieve what isessentially a constant rate of release of the active compound for arelatively long period of time, e.g. 1 week or more, both in connectionwith local applications and in connection with systemic applications.

Furthermore, the composition is also suitable for other knownpharmaceutical uses of heparin, including both systemic and local uses,e.g. for inhibiting blood coagulation. In addition, surgical methods canalso be used to administer the composition in the form of an implant.

It was possible to administer the preparation subcutaneously topatients, for the purpose of thrombosis prophylaxis, in a manner whichwas to a large extent pain-free. The administration was repeated severaltimes at intervals of 7 days.

The hyaluronic acid can be used in uncrosslinked or crosslinked, e.g.covalently or noncovalently crosslinked, form. The crosslinkedhyaluronic acid can be prepared in a manner which is known per se. Inthis connection, the covalent crosslinking is in general effected bycrosslinking with bifunctional reactive agents, such as glutaraldehydeor carbodiimide; by way of bifunctional amino acids, e.g. lysine,protamines or albumins. However, it is also possible, for example, forcrosslinkings to be produced by way of an amide bond. Other reagentswhich are suitable for covalently crosslinking hyaluronic acid areethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether,divinylsulfone, photocrosslinking reagents, such as ethyl eosin, andhydrazides, such as bishydrazide compounds, trishyrazide compounds andpolyvalent hydrazide compounds. It is furthermore also possible to usehyaluronic acid derivatives which are esterified intramolecularly and/orintermolecularly.

Particular preference is given to crosslinking in a noncovalent manner,using multiply charged metal ions, such as iron, copper, zinc, calcium,magnesium, barium and other chelating metal ions.

Hyaluronic acid is commercially available in the crosslinked state (e.g.Hylon® and Hylagel®, a crosslinked hyaluronic acid from Biomatrix, N.J.,USA; for the preparation cf. U.S. Pat. No. 4,713,448 and U.S. Pat. No.4,605,691 as well, APC® from Fidia, Incert® from Anika Therapeutics orIntergel® from LifeCore) and can then be used in accordance with theinvention after having been associated with heparin.

In a particularly preferred embodiment, long-chain hyaluronic acid(molecular weight preferably between 10⁴ and 10⁶ Da, in particularbetween 10⁵ and 10⁶ Da) is used; it is then possible for the degree ofcrosslinking to remain low. Short-chain hyaluronic acid is also suitablewhen the degree of crosslinking is higher, with it also being possibleto use molecules having a low chain length of only a few, e.g. ≧10,preferably ≧20, saccharide units.

The association of the heparin with the hyaluronic acid can be effectedcovalently or noncovalently, e.g. by means of chemical crosslinking orby means of chelate formation, as explained above. Physiologicallytolerated multiply charged metal ions, such as Mg²⁺, Ca²⁺, Zn²⁺, Fe²⁺ orFe³⁺, are preferably used for the chelate formation.

The pharmaceutical compositions according to the invention preferablycomprise the hyaluronic acid in quantities of from 0.01 to 20% byweight, based on the total pharmaceutical composition, in particular ina quantity of from 0.01 to 5% by weight and, particularly preferably, ina quantity of from 0.01 to 1% by weight.

The proportion of heparin in the compositions can be varied within wideranges and depends on the size and nature (e.g. crosslinked anduncrosslinked) of the heparin and its association with the hyaluronicacid and the envisaged nature and duration of the application. Ingeneral, the proportion is in the range of from 0.1 to 20% by weight,based on the total pharmaceutical composition, in particular of from 0.5to 10% by weight, and particularly preferably of from 1 to 5% by weight.Depending on the application, the heparin can be present in long-chainor short-chain, crosslinked or uncrosslinked form. Preference is givento using short-chain heparin having a size of 5-50, in particular 5-10,saccharide units.

The pharmaceutical compositions according to the invention can bepresent in the form of preparations which can be administered by meansof injection or surgical interventions and, in particular, in the formof injectable or implantable gels or solutions, preferably having awater content of from 60 to 99% by weight, or else as an anhydrousprecursor, e.g. lyophilized powder in pulverulent form. Thepharmaceutical auxiliary and carrier substances which can be used forthis purpose are customary substances which are suitable for theapplication according to the invention and which are compatible withhyaluronic acid and heparin. The preferred carrier substance is water oran aqueous buffer solution.

Examples of pharmaceutical auxiliary substances which the pharmaceuticalcompositions according to the invention can comprise are agents foradjusting the pH, stabilizing agents, antioxidants, solubilizers,penetration-promoting agents, preservatives and/or gelatinizing agents,as are customarily used in compositions of this nature. They areemployed in the quantities which are customary in preparations of thisnature.

In addition to the true active compounds hyaluronic acid plus heparin,the pharmaceutical compositions according to the invention can, whereappropriate, also comprise additional pharmaceutical active compoundswhich are compatible with the hyaluronic acid and the heparin within thecontext of the application, e.g. active compounds for treating skindiseases (dermatoses), antibiotics, e.g. gentamycin, vancomycin,penicillins or cephalosporins, sulfonamides, disinfectants, hormones(e.g. corticoids) and hormone derivatives (e.g. cortisol), localanesthetics, e.g. of the lidocaine or novocaine type, vasoactivesubstances for vascular constriction (avoidance of hemorrhages),adrenaline, enzymes, such as hyaluronidase, interleukins, growthfactors, e.g. EGF, PDGF or IGF, skin care agents and/or bloodflow-promoting (hyperemizing) agents. The additional active compoundscan, where appropriate, be associated with the hyaluronic acid, e.g. bymeans of covalent or noncovalent interactions.

In a preferred application according to the invention in the form of aninjection, the preparations can comprise local anesthetics, e.g. for thepurpose of avoiding pain when puncturing with the injection needle.

The compositions according to the invention can be produced in awell-known manner which is customary per se for producing compositionsof this nature. In this connection, the sequence in which the individualconstituents are mixed is as a rule not critical.

The nature, dose and frequency of the administration of the compositionaccording to the invention, as well as its constitution (e.g. viscosity,degree of crosslinking, content of active compound, etc.) depend, inparticular, on the nature and severity of the disease as well as on thestate of health of the patient and the condition of the site ofadministration, e.g. the condition and the sensitivity of a scar and ofthe skin or of a wound following a surgical intervention. If thecompositions according to the invention are administered in the form ofpreparations which can be applied topically, the administration then asa rule conforms with the conditions which are customary for compositionsof this nature.

The nature of the treatment, and the frequency of the application, alsodepend, in particular, on the individual response of the persons to betreated. Gels or solutions are preferably applied at intervals of fromseveral days up to one or two months, in particular of from approx oneto two weeks.

If the compositions according to the invention are administeredintralesionally in the form of injectable gels, this then preferablytakes place by injection with the aid of fine needles and usingcompression-resistant syringes. However, the gels according to theinvention can also be shot in transdermally using pressure devices;pressure devices as are known in medicine for such administration can beused for this purpose. Certain preparations can also be administeredsystemically, i.e. into the blood circulation or into body cavities, forexample following surgical intervention. Implantable compositions arepreferably present in the form of viscous gels or solutions, i.e. whatare termed instillation solutions.

As a result of its association with long-chain hyaluronic acid,uncrosslinked, and even short-chain, heparin can be administered, as anactive compound, by injection. The compositions according to theinvention prevent the rapid removal of the heparin from the site ofaction which would otherwise take place in the absence of itsassociation with hyaluronic acid. Depending on the breakdown of thehyaluronic acid matrix, and on the nature of its bonding, the heparinremains active at the site of administration, e.g. in the keloid tissue,for days, weeks or months. Particular preference is given to theduration of action being 5-20 days, e.g. approx. 1-2 weeks.

An advantage of the preferred preparations according to the invention,e.g. in the form of injectable or implantable gels and theiradministration, is also that no additional hygienic measures whatsoeverare required after the injection sites or the surgical sutures havehealed. It is possible to treat all body regions equally and themobility of the patient is not restricted by dressings. Treatment withthe preparations according to the invention can prevent the appearanceor reappearance of keloids, thereby demonstrating the prophylacticeffect of these preparations.

Another application form for preventing keloids or contracted scars isthat of administering anhydrous compositions (e.g. as a lyophilisate) tofresh wounds or body cavities in the form of wound powder. In thisconnection, the powder is sprinkled into the open wound or wound cavitybefore the wound is closed. The wound is then closed by means ofsutures, clips or the like. In the wound, the powder absorbs water fromthe tissue, and then corresponds to the preparation according to theinvention in the form of a gel, or itself constitutes a preparationaccording to the invention.

In order to prevent unwanted adhesions, compositions in powder form orgel form can also be introduced into large body cavities, for exampleinto the abdominal cavity or thoracic cavity, during a surgicaloperation on the intestine or on the lung, into the pericardium or byway of indwelling drainages following surgical operations. In the caseof inflammatory discharges into large body cavities, the preparationaccording to the invention can also be introduced by way of theindwelling cannula following puncture and evacuation of the discharge.

The preparation according to the invention can also be introduced intoexternally accessible cavities and passageways of the body, for exampleinto the main nasal cavities and paranasal sinuses or nasal meatus, orinto the tear ducts, for the purpose of preventing scarred adhesions,possibly on a suitable support (e.g. tampon) as well.

Novel applications of pharmaceutical compositions which comprisecrosslinked or uncrosslinked hyaluronic acid as active compoundconstitute yet another aspect of the invention. The reader is referredto the above comments with regard to the hyaluronic acid content ofthese compositions and their administration forms.

In a first aspect, these compositions are intended for treating viralinfections, for example in connection with treating infections withneurotropic viruses, such as herpesviruses, e.g. herpes simplex orherpes zoster. The compositions can be administered locally orsystemically depending on the nature of the viral infection. Thecomposition is preferably used for treating dermal or mucosal herpesinfections, for example herpes labialis or herpes genitalis. Otherpreferred areas of application are the treatment of infections withhepatotropic viruses, such as hepatitis viruses, e.g. hepatitis A, B orC viruses, the treatment of infections with immunotropic viruses, e.g.HIV or cytomegalovirus, or the treatment of infections with otherneutrotropic viruses, e.g. polio. It is furthermore also possible totreat infections with other viruses which cause diseases of the eyes,e.g. epidemic keratoconjunctivitis, or of the airways, e.g. colds, nasalcatarrh or influenza. Examples of these viruses are rhinoviruses andinfluenza viruses. In some cases, a local, e.g. topical or transdermaladministration is preferred. Other preferred administration forms areopthalmological compositions or compositions for nasal administrations,e.g. drops, sprays and inhalable aerosols. On the other hand, thecomposition can also be administered systemically, e.g. by means ofintravenous injection, or orally as a solution for drinking or rinsing,e.g. for controlling infections of the gastric and intestinal tract orof the thorax. Particular preference is given to a single or repeatedprophylactic administration prior to the onset of an acute disease.

Surprisingly, it was found that, when the composition in the form of agel was administered by intradermal or subdermal injection into theaffected region in patients suffering from recurring herpes labialis, itwas possible, at least to a large extent, to prevent the onset of theinfection. After the compositions had been administered, the patientswere free from skin eruptions. Administration in the prodromal stage wasparticularly effective in connection with the onset of pruritus. Whereappropriate, the administration of the composition prophylactically canbe repeated at relatively large time intervals, for example of 3 months.

Another application of the hyaluronic acid-comprising compositions isthat of using them as analgesics, preferably as analgesics which actperipherally, in particular for administration in the region of nerveendings, e.g. of injured nerve endings, for example following incisionwounds. In this connection, the administration can, for example, beeffected locally by injection, as previously described. In severalcases, administration of the composition into surgical wounds, forexample as a powder, gel or solution prior to wound closure, resulted inconsiderable pain alleviation and also in a substantial decrease in therequirement for additional peripheral or central analgesics.

Yet another application of the hyaluronic acid-comprising compositionsis that of tautening skin. Surprisingly, it was found that, when thecompositions were administered, it was possible to achieve a discernibleeffect which was long-lasting, i.e. which lasted over a period of atleast six months and, in particular, over a period of from one toseveral years. Thus, the forehead wrinkles of several patients werevisibly reduced one year after administering the composition. Thecompositions are consequently suitable for use as “lift serum” fortautening facial skin and can contribute to avoiding surgicalinterventions for the purpose of facial tautening. The administration ispreferably transdermal, e.g. by means of injection or by using one ofthe previously described transdermal administration systems, and can beeffected in a punctate manner and/or extensively on the skin regions tobe tautened.

Hyaluronic acid can also be added to infiltration solutions which areinjected into the tissue prior to a liposuction. These infiltrationsolutions are usually isotonic or hypotonic salt solutions which cancontain additives, for example blood vessel-contracting and/orpain-reducing additives, such as noradrenaline, adrenaline, lidocaine,prilocalne, bicarbonate, corticosteroids, etc. Hyaluronic acid(crosslinked and/or uncrosslinked hyaluronic acid) is advantageouslyadded to these solutions at concentrations of from 0.001 to 1.0%(wt/vol), preferably of from 0.01 to 0.5% (wt/vol), e.g. of about 0.025%(wt/vol). It was found that adding the hyaluronic acid mechanicallyfacilitates the process of liposuction, i.e. manipulating the suctioncurette, which is moved through the adipose tissue, requires less energyinput. Furthermore, structures in the adipose tissue which are worthconserving, such as blood vessels, nerves or connective tissue bands,are spared. Adding the hyaluronic acid also makes the process ofliposuction markedly more atraumatic when compared with the conventionalprocedure. Finally, adding the hyaluronic acid to the infiltrationsolution improves the flow properties of the adipose tissue aspiratesuch that it is possible to use a lower negative pressure. This alsoleads to a reduction in the tissue trauma. All in all, adding hyaluronicacid to the infiltration solution protects the remaining adipose tissuein the body and results in the adipose tissue aspirate being withdrawnin a gentler manner. This thereby improves the survival of fat cells inthe aspirate.

This is of importance insofar as adipose tissue aspirates can be used asendogenous transplants. Adding hyaluronic acid to the infiltrationsolution protects the fat cells during withdrawal such that, whentransplanted, the transferred tissue is seen to grow more strongly atthe recipient site. As a consequence, hyaluronic acid is alwaysparticularly preferably added to the infiltration solution when theaspirated adipose tissue is to be transferred to other sites of the bodyas an autologous transplant. The invention consequently relates to aadipose tissue aspirate which contains hyaluronic acid (crosslinked oruncrosslinked).

Particular preference is given to using the fat aspirate for cosmeticpurposes, e.g. for upholstering soft parts in the face. In this case,the aspirate is inserted into the recipient site through thin cannulaehaving, for example, a diameter of 1-2 mm. The presence of hyaluronicacid in the aspirate or transplant results in the passage of the tissuethrough the cannula being facilitated. This means less application ofpressure for effecting transport through the cannula, lesstraumatization of the transferred tissue and/or a higher rate of growthof the transferred tissue at the recipient site.

The hyaluronic acid-containing adipose tissue aspirate according to theinvention can also be added, after conventional aspiration (without anyaddition of hyaluronic acid), at relatively high concentration, e.g.0.1-1% (wt/vol), to the aspirate adipose tissue moiety (fraction) to betransferred, with this likewise resulting in the rate of take of thetransferred tissue being increased.

In general, when, for example, transplants of autologous or xenogenictissues and/or autologous or xenogenic loose or isolated cells, whichare not consolidated into a tissue formation, are being transferredthrough cannula-shaped applicators, an addition of hyaluronic acid(crosslinked and/or uncrosslinked) can improve the survival and/or thegrowth of the transferred tissue particles or cells.

It is already known that cells which are subsequently intended to betransferred into a recipient organism are treated with hyaluronic acidin a culture. However, it has been found that the concentration ofhyaluronic acid in these cultures is not sufficient to ensure aprotective effect when the transfer takes place through thin cannulae.It is therefore advisable, before transferring such cells, to effect anaddition of hyaluronic acid in accordance with the invention, with thefinal concentration of hyaluronic acid advantageously being adjusted tofrom 0.05 to 1.0%, in particular from 0.1 to 0.5% (wt/vol).

Insofar as tissue particles or cells are being transferred into bodycavities, hyaluronic acid additionally acts as a lubricant andconsequently also as a protective film for transplants, for example inthe pericardium, in the pleural cleft or in the peritoneum, since organsare moved against each other in these cavities or cleft spaces.Hyaluronic acid is consequently finally also to be used as a lubricantin the abovementioned cleft spaces.

1. A method for treating influenza or influenza viral infections of therespiratory system in a patient, comprising the steps of: (a) providinga pharmaceutical composition comprising crosslinked or uncrosslinkedhyaluronic acid and a customary pharmaceutical auxiliary and/or carriersubstance, wherein the crosslinked or uncrosslinked hyaluronic acidserves as the only active ingredient in the pharmaceutical composition;and (b) administering the pharmaceutical composition in the form of aspray or an inhalable aerosol by means of therapeutic interventions.